All of the publications, patents and patent applications cited within this application are herein incorporated by reference in their entirety to the same extent as if the disclosure of each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.
There is an increasing demand for a small scale array-based and/or microfluidic device that processes micro- or nano-volumes of sample, with time and cost savings arising from miniaturization. Prior art approaches to miniaturised polymerized chain reactions (“PCR”) make use of open or enclosed chambers or flow through zones/channel networks with appropriate temperature regulation; some have on-board silicone rubber-based or magnetic-based valving and/or pumping. Although potentially powerful approaches, challenges may arise of pressure seal and/or evaporation, pressure buffering, as well as others such as chemical interference through surface interactions, and evaporation/contamination via the porous, gas permeable membranes used in pumps and valves.
Performing PCR in a colloidal hydrogel matrix (hereafter termed “gel”) may confer a multitude of advantages. For example, the DNA, polymerase enzyme and other PCR reagents a) have reduced access to the device materials' surfaces where they may be adsorbed, absorbed, poisoned or otherwise rendered inactive and b) are kept within close proximity to each other without the need for valves or pumps. Likewise, any contaminant solutes from device materials have reduced access to the PCR reaction.
Gels provide a successful medium for PCR, as first introduced by Chetverin et al., see for example U.S. Pat. No. 5,616,478; which is herein incorporated by reference in its entirety. PCR was confined to circular spots in a gel sheet where the initial DNA or RNA templates, formed “molecular polonies” (short for polymerase colonies), named for their similarity to the growth of bacterial colonies in agar; the initial amount of DNA can be accurately estimated by counting the number of polonies. Mitra et al. (Mitra, R. D. et al; Nucleic Acid Research 1999, 27, e34) performed DNA amplification in a thin acrylamide film polymerized with all the reagents along with plasmid DNA as their template. In an alternate approach, Strizhkov et al. (Strizhkov, B. N. Et al; Biotechniques 2000, 29, 844-857) used nanoliter gel pads to immobilize primers for PCR. Single Nucleotide Polymorphisms (SNPs) in cDNA were detected with polony technology by Butz et al. (Strizhkov, B. N. et al; 2000, 29, 844-857)
Absent the use of immobilized primers within the gel, previous instances of in-gel PCRs were performed in a defined chamber with relatively large volumes (62-65 μL). The present art is in need of a means to perform post PCR analysis of amplicons, such as melting curve analysis (“MCA”) without imposing additional steps for the transfer or handling of PCR product and in small volumes normally associated with microfluidic reactions (sub 100 μl).